Viola belonging to the family Violaceae is a genus of mostly perennial herbs. They are almost cosmopolitan but mostly distributed in the temperate zones and restricted to the mountains in the tropics (Anonymous, 1965, Indian Pharmacopoeia, 2nd Edn. Govt. of India; Anonymous, 1976, Wealth of India, PID, CSIR, New Delhi, Vol. X: 514-517). There are about 400 species of Viola, of which about 30 species are found in India. Viola species commonly known as “violets” or “pansies” hybridize freely in nature and, therefore, are difficult to be distinguished taxonomically (Anonymous, 1976, Wealth of India, PID, CSIR, New Delhi, Vol. X: 514-517). The plants are available as different cyto types with 2n=20, 37, 54 etc. in nature (Canne, J. M., 1987, Canad J. Bot., 65; 653-655). Most of the Viola species yield perfumes and medicaments, while some are ornamentals (Woodland, D. W., 1991, Contemporary plant systematics, Prentice Hall, Eaglewood, Cliffs). The most important among these species, which is known for its medicinal significance in indigenous system of medicine, is Viola odorata L., which is considered to constitute the genuine drug called “Banafsha” (Handa, K. L., Kapoor, L. D., Chopra, I. C. and Nath, S., 1951, Indian J. Pharmacy, 13: 29-48). The whole plant is used in medicine and is available usually in 3 forms: 1) the dried aerial parts of the herb i.e. the stem, leaves and flowers; 2) only dried flowers and 3) aerial parts without flowers. The plant is valued as sedative, expectorant, diaphoretic, antipyretic, diuretic, emetic, purgative, hypotensive and as a laxative (Kirtikar, K. R. and Basu, B. D., 1933, Indian Medicinal Plants, I: 205-212, Lalit Mohan Basu, Allahabad; Chopra, R. N., Nayer, S. L. and Chopra, I. C., 1956, Glossary of Indian Medicinal Plants, CSIR, New Delhi). It has also been reported to be antimycotic and antibacterial and is used for the treatment of eczema and inflammation. The flowers are credited with emollient and demulcent properties and are used as remedy for cough, sore throat, hoarseness, ailments of infants, billiousness and lung troubles and are also listed in French Pharmacopoeia (Lamaison, J. L., Petitjean, C. F. and Carnet, A., 1991, Plantes-Medicinales—et Phytotherapie, 25: 79-88). In Unani system of medicine, this plant is the main ingredient of “Joshanda” consisting of mixtures of drugs and it is mostly used for cold, catarrhal, cough, and associated fevers (Khetrapal, K., Khanna, T., Arora, R. B. and Siddiqui, H. H., 1987, Indian J. Pharmacy, 19: 200-204). The drug is prescribed mainly in the form of decoction, jam or syrup.
In India, Viola odorata wildly grows mainly in Jammu and Kashmir and often cultivated elsewhere particularly in West Bengal, Gujarat, Andhra Pradesh, Karnataka and Tamilnadu (Sharma, B. D., Balkrishan, N. P. 1993, Flora of India 2: 351-379). Due to the limited distribution and high demand of Viola odorata, which is the main source of banafsha, alternate species including V. betonicifolia, V. biflora, Viola canescens, V. pilosa, V. sylvestris and V. tricolor (some of which grow abundantly through out the hilly regions of India) have been reported to be supplied in the market as banafsha, either in the pure form or after mixing with V. odorata (Handa, K. L., Kapoor, L. D., Chopra, I. C. and Nath, S., 1951, Indian J. Pharmacy, 13: 29-48: Dhar, et al, 1968, Indian J. Exp. Biol., 6: 245; Mehrotra, S., Rawat, A. K. S. and Shome, U., 1998, Natural Plant Sci., 4: 14-22) which have led to the questionable efficacy of the drug, undermining the importance of Viola in medicine as originally defined (Kirtikar, K. R. and Basu, B. D., 1933, Indian Medicinal Plants, I; 205-212, Lalit Mohan Basu, Allahabad; Handa, K. L., Kapoor, L. D., Chopra, I. C. and Nath, S., 1951, Indian J. Pharmacy, 13: 29-48). It has also been reported that the roots of Viola odorata, V. cinerea and V. tricolor, which are emetic in nature, are also used as substitute or adulterants of ipecac drug, which originally comes from dried roots and rhizomes of Cephaelis ipecacuanha of family Rubiaceae (Ananymous, 1976, Wealth of India, PID, CSIR, New Delhi, Vol X: 514-517; Chopra, R. N., Nyer, S. L. and Chopra, I. C. Glossary of Indian Medicinal Plants, 1956, Edn. CSIR, New Delhi, 256). It is imperative, therefore, to identify the species composition of banafsha and other herbal medicines before using them in various medicinal preparations, specially by the industrial houses. The classical ways of identifying herbal medicines are based on the morphological, anatomical and chemical analysis using a variety of modern tools (Thankamma, A., Radhika, L. G., 1997, Aryavaidyan, XI: 52-56; Chauhan, S. K., Singh, B. P., Agarwal, S., 1995, Indian Drugs, 36: 189-191; Gawan, S. and Grampurohit, N. D., 1999, Indian Drugs, 36: 175-180). However, the structures and chemical profiles of the medicinal herbs are effected during sample processing and also by environmental and developmental factors during plant growth (Li, P., Pu, Z. M., Jiang, X., Liu, H. J., Xu, G. J., 1994, J. Plant Res. Envir., 3: 60-63; Cai, Z. H., Li, P., Dong, T. T. X. and Tsim, K. W. W., 1999, Planta Medica, 65: 360-364), for example, where market samples are in dry and powdered form, it is difficult to identify plant species by morphological characters. Similarly age, origin, harvesting period and the method of drying of plants may lead to the differences in the secondary product formation and may interfere in identification based on chemical analysis.
Molecular markers are helpful in such situations, since they are independent of environmental and developmental factors. They can be obtained even from dried and powdered herbs. RAPD markers have been used to distinguish 3 different series of Viola tricolor (Oh, B. J., Ko, M. K., and Lee, C. H., 1998, Plant Breeding, 117: 295-296), to detect individual component of Chinese medicinal prescription (Cheng, K., Tsay, H., Chen, C. and Chou, T., 1998, Planta Medica, 64: 563-565) and to detect dried roots of 3 Paax species and their adulterants (Shaw, P. C. and But, P. P. H., 1995, Planta Medica, 61: 466-469). However, these markers are of little value specially in freely hybridizing populations including Viola. Conserved sequences in such cases could be ideal. In fact, rRNA genes and their associated spacer length variability has been utilized to differentiate fungal species (Nazar, R. N., Hu, X., Schmidt, J., Culham, D. and Robb, J., 1991, Physiol. and Mol. Plant Pathol., 39: 1-11; Robb, J., Moukhamedov, R., Hu, X., plate, H. and Nazar, R. N., 1993, Physiol. and Mol. Plant Pathol. 43: 423-36; Moukhamedov, R. S., Hu, X., Nazar, R. N. and Robb, J., 1994, Phytopathology, 83: 256-259), to identify plant varieties and species (Martsinkovskaya, A. I., Moukhamedov, R. S. and Abdukarimov, A. A., 1996, Plant Mol. Biol. Reportr. 14: 44-49) and also to detect cereal composition in admixtures (Ko, H. L. and Henry, R. J., 1996, Plant Mol Biol. Reportr, 14: 33-43). Here, the applicants describe cloning and sequencing of the spacer regions between 5S rRNA genes in five Viola species, utilization of the sequence differences to detect the individual Viola species by a simple polymerase chain reaction and to utilize this technology for authentication of commercial samples of “banafsha”.